A light-scattering study of bovine factor VIII

Classical light-scattering measurements made on bovine Factor VIII preparations, which contain a series of multimers ranging from 1 greater than 12 X 10(6) molecular weight, gave a weight average molecular weight of about 8 X 10(6) and a z-average radius of gyration of about 3000 A. Characteristic dimensions for variously shaped models were calculated.     

Comparison of Ca uptake characteristics of microsomal fractions isolated from unfertilized and fertilized sea urchin eggs

The microsomal fraction isolated from sea urchin H. pulcherrimus eggs has the ability to actively accumulate Ca²⁺ in the presence of ATP. The Ca²⁺ uptake was sustained by addition of oxalate and was apparently insensitive to sodium azide. The sequestered microsomal Ca was readily released by the divalent cation ionophore A23187. The microsomal fraction obtained from fertilized eggs accumulated Ca²⁺ about five times more quickly than did that from unfertilized eggs. The increased Ca²⁺ uptake by microsomal fraction obtained from fertilized eggs was due to an increase in the maximum velocity of Ca²⁺ uptake and there was no difference in K_m for calcium between the two fractions.     

Pyruvate kinase isozymic shifts of differentiating chick myogenic cells in vivo and in culture.

Developing chick skeletal muscle undergoes an isozymic shift from type K pyruvate kinase to type M during development. A major increase in pyruvate kinase activity follows the isozymic shift, resulting in at least 40-fold higher specific activities by adulthood. Similar isozymic changes occur in primary cultures of myogenic cells from 12-day-old chick embryos. Cultures initially contain only type K pyruvate kinase. Type M appears by the fourth day of culture and accounts for 80–90% of the activity by the eleventh day. Type M did not accumulate when cell fusion was prevented by removing Ca²⁺ from the growth medium or when protein synthesis was inhibited by cycloheximide.     

Cyclic AMP potentiates down regulation of epidermal growth factor receptors by platelet-derived growth factor

Pretreatment of $\text{Balb}\text{c}\text{-3T3}$ cells with platelet-derived growth factor (PDGF) has been shown to decrease binding sites for ¹²⁵I-labelled epidermal growth factor (EGF) (1,2,3). Agents which elevate cellular cyclic AMP concentrations enhance this ability, and the change in EGF binding is inversely proportional to the elevation of cyclic AMP. In quiescent density arrested cells, the sensitivity of cells to down regulation of EGF receptors by PDGF is proportional to the cyclic AMP content of the cultures in three different cell lines. Agents which elevate cyclic AMP and which potentiate PDGF mediated heterologous down regulation of EGF receptors are able, like cholera toxin (3), to stimulate cells to synthesize DNA in defined medium in the absence of EGF. Down regulation of EGF receptors by PDGF in combination with agents elevating cyclic AMP effectively mimics the action of EGF.     

Rapid colorimetric assay for intestinal peptide hydrolases

A simple two-step method is described for quantitating the release of free l-phenylalanine, l-leucine, l-methionine, or l-isoleucine from di- or polypeptides. The colorimetric assay is based on the ability of l-amino acid oxidase to catalyze the oxidation of free l-amino acid, but not of peptides. The potent metal chelator 1,10-phenanthroline, which is included in the second step of the assay, effectively inhibits peptide hydrolase activity thus permitting the assay to be carried out in two sequential steps in the same test tube with no intervening enzyme-destroying step. Although the assay is indirect and subject to interference by some chemicals, it is not affected by a large number of compounds frequently used in enzyme studies. The method was used to study the subcellular distribution of a number of peptide hydrolase activities in rat intestinal mucosa. For nine substrates, more than 80% of the total recovered activity was present in the cytoplasmic fraction while for two substrates, phenylalanylglycine and phenylalanylglycylglycine, more than 60% of the activity recovered was present in the particulate fraction.     

Isolation and sequence determination of a peptide located in or near the active site of bovine muscle pyruvate kinase

Bovine muscle pyruvate kinase was inactivated by treatment with trinitrobenzenesulfonic acid; approximately one trinitrophenyl group was incorporated per subunit. ADP or Mg-ADP decreased the rate of inactivation but Mg⁺⁺ alone or phosphoenolpyruvate had no effect. The inactivated protein was treated with trypsin and the trinitrophenylated peptide isolated by gel filtration. Homogeneity of the isolated peptide was shown by high voltage electrophoresis and high pressure liquid chromatography. Amino acid analysis and sequence determination revealed the presence of an acidic peptide 34 amino acids long and containing ?-trinitrophenylated lysine.     

pH dependence of magnesium ion binding to prothrombin fragment 1 and gamma-carboxyglutamic acid-containing peptides via 25Mg NMR.

The binding of magnesium ions to two tripeptides, $\text{L}$-Arg-$\text{D}$-Gla-$\text{D}$-Gla-OMe and Z-$\text{L}$-Arg(NO₂)-$\text{D}$-Gla-$\text{D}$-Gla-OMe, and to bovine prothrombin fragment 1 as a function of pH has been monitored by ²⁵Mg NMR spectroscopy. Binding to the tripeptide was dependent on peptide ionizations occurring at pH 4.6 – 4.8. The pH dependence of magnesium ion binding to fragment 1 reveals two inflection points 4.2 may be attributed to the deprotonation of the third side chain carboxylic acid group of the double γ-carboxyglutamic acid sequence. The origin of the increased binding of magnesium ions to fragment 1 at pH values above 7 is unknown.     

Isolation of polypeptides containing the intermolecular cross-link , '-dihydroxylysinonorleucine from dentin collagen

Insoluble dentin collagen was reduced with sodium borotritiide and then sequentially cleaved with cyanogen bromide and trypsin. Separation and purification of the labeled peptides were accomplished by gel filtration and ion-exchange chromatography. Two peptides were obtained containing 38 and 26 residues each, respectively. Both contained stoichiometric amounts of the collagen intermolecular cross-link δ,δ′-dihydroxylysinonorleucine. Their compositions are reported. The data indicate that one of the branches on each of the H-shaped peptides might be identical and the other branch on each is derived from different loci on the collagen molecule. Neither crosslink peptide involves the N-terminal portion of collagen.     

Opioid peptides and catecholamines in human pheochromocytoma

Opioid peptides (OP) and catecholamines (CA) were measured in twelve human pheochromocytomas (PHEO). In all tumors the CA concentrations were much higher than those OP (range: 300-85,000 fold higher). Large intertumor variability in the levels of both substances was encountered (mean +/- S.E.M. = CA: 44.9 +/- 7.7 mumoles/g; OP: 38.1 +/- 17.1 nmoles/g; range = CA: 10.1-93.1 mumoles/g; OP: 0.7-181 nmoles/g). Norepinephrine (NE) was the main CA in seven of the 12 tumors. In four of these PHEO, NE accounted for 85% or more of the total CA. These "noradrenergic PHEO" derived from the right adrenals, were of smaller size (36 +/- 15g), had the lowest levels of OP (1.1 +/- 0.3 nmoles/g) and CA (28 +/- 10 mumoles/g), produced moderate to severe sustained hypertension (MBP: 160 +/- 11 mmHg) and the most severe and persistent clinical manifestations. Epinephrine (EPI) was the main CA in five of the 12 tumors. These PHEO had intermediate levels of OP (12 +/- 3 nmoles/g), and four of them were of left adrenal origin. Patients bearing these tumors were generally normotensive (MBP: 103 +/- 4 mmHg) and asymptomatic, with occasional paroxysmal crisis. The highest levels of OP (132 +/- 24 nmoles/g) were found in two tumors of extra-adrenal location and in one of right adrenal origin. The proportion of NE and EPI ranged between 60-80% and 20-40% respectively, of total tumor CA. The two extra-adrenal PHEO were the largest of this series (180 and 245g). These patients had mild hypertension (MBP: 118 +/- 7 mmHg) of sustained or paroxysmal course, and frequent symptomatic episodes. Differences in the synthesis, storage, metabolism and release of CA and OP in PHEO probably account for their variable tumoral content, as well as for the clinical heterogeneity produced by these tumors. It remains to be seen whether OP can contribute to the clinical manifestations of patients with pheochromocytomas.     

Regulation of insect prothoracic glands: Existence of a haemolymph stimulatory factor in Manduca sexta

The primary regulator of ecdysone biosynthesis by insect prothoracic glands is the prothoracicotropic hormone. However, it now appears that other factors, secondary regulators, may modulate prothoracic gland activity. One such factor has been isolated from the haemolymph of Manduca larvae. This haemolymph factor stimulates in vitro ecdysone synthesis by larval and pupal prothoracic glands by approx. 5-fold. It has an apparent mol. wt of ～330 kD, is protease-sensitive and is heat labile, the latter clearly distinguishing it from the prothoracicotropic hormone. Further, its steroidogenic effects and those of prothoracicotropic hormone are additive. Treatment of larval or pupal prothoracic glands with both moieties simultaneously effects an approx. 10-fold increase in ecdysone synthesis. The haemolymph titre of the stimulatory factor is low at commitment of the last-larval instar, then increases by approx. 3-fold later in the instar during pharate-pupal development. This increase in the titre is sufficient to effect a significant increase in prothoracic gland activity that could be physiologically important. Thus, it appears that the fluctuating level of this haemolymph stimulatory factor may act in conjunction with prothoracicotropic hormone to regulate the haemolymph ecdysteroid titre by modulating the ecdysone biosynthetic activity of the prothoracic glands.     

Characterization of a lymphokine produced by human T cells which inhibits collagen synthesis

Human lymphocytes, isolated from peripheral blood, were cultured for 48 hr in a defined medium containing 10 mg/ml bovine serum albumin and phytohemagglutinin. A lymphokine which inhibits collagen synthesis by cultured human dermal fibroblasts was purified from the lymphocyte incubation medium by successive steps of ammonium sulfate precipitation, gel filtration chromatography, and isoelectric focusing. Good recovery of this collagen synthesis inhibitory factor (CSIF) was obtained and a factor with an approximate molecular weight of 55,000 and a pI of 6.2 was isolated. The purification of the factor should permit further studies on its mechanism of action.     

Energy-dependent endocytosis in erythrocyte ghosts. 3. Hydrophobic character of maleimide inactivation sites of ATPase and of endocytosis

The effects of a variety of reactive compounds on endocytosis in erythrocyte ghosts were observed. Of these reagents, only alkylating reagents were effective at low concentrations. This suggested that an alkylatable site, probably a sulfhydryl group, was important in endocytosis. In a series of N -substituted maleimides, effectiveness of the alkylating agent in inactivating both ATPase and endocytosis correlated very well with a high value of the partition coefficient between octanol and water. This suggested that a hydrophobic region was present at the site of inactivation, so that strongly hydrophobic alkylating agents were bound more firmly by this site. The action of the N -substituted maleimides was clearly due to the reactivity of the carbon-carbon double bond in the heterocyclic ring, since saturation of this bond completely destroyed the effectiveness of the inhibitor. Statistical analysis of the dependence of the effectiveness of N-substituted maleimides upon partition coefficient and Hammett sigma parameters showed that the partition coefficient was by far the most important factor which controlled the effectiveness of these inhibitors. The sigma parameter played a lesser role. The dependence of the effectiveness of the maleimides on these two parameters was the same, within the statistical error, for both the ATPase activity and endocytosis activity. This suggested that inhibition of endocytosis was due to reaction with the same site responsible for inhibition of ATPase.     

Epidermal growth factor (EGF) is required only during the traverse of early G1 in PDGF stimulated density-arrested BALB/c-3T3 cells

Density-arrested BALB/c-3T3 cells that had received a transient exposure to PDGF and were then transferred to medium containing only EGF and somatomedin C (Sm-C) began DNA synthesis after the G0/G1 lag. Supraphysiological concentrations of insulin could be employed to replace the Sm-C requirement. This G0/G1 lag phase was bisected by the requirement for the exogenous presence of EGF. Our data indicated that EGF was required during the traverse of only the first half of G0/G1 phase (6 h) and not during the traverse of late G1. Subphysiological serum concentrations of Sm-C were also necessary to be present with EGF for progression through early G0/G1; however, traverse of the final half of G0/G1 and commitment to DNA synthesis required the presence of Sm-C. It was found that physiological concentrations of Sm-C were required for the traverse late G1. The requirement for Sm-C for G0/G1 traverse of BALB/c-3T3 cells as opposed to human fibroblasts or glial cells may be due to a difference in endogenous synthesis of an insulin-like growth factor. Our data are in close agreement with previous reports that EGF is only required for approximately the first 8 h during traverse of the G0/G1 phase. The requirement for EGF to be present for the first 6 h of G0/G1 could result from a continued or repetitious event or by more than one distinct EGF-requiring event.     

Influences of cell volume and adrenalectomy on cation flux in dog red blood cells

Rates of ²⁴Na and ⁴²K entry into dog red blood cells were found to be strongly influenced by cell volume. The kinetics of isotope movement were complex, and the cells were not in a steady state. By applying a simple, two-compartment equation to the early times points, values for flux were calculated and corrected for the changes in surface/volume ratio which occur when cells are shrunken or swollen. Curves were thus generated showing Na and K influx as functions of cell water content. A reinvestigation of the effects of adrenalectomy showed that all the observd changes in Na flux could be explained on the basis of alterations in red cell volume.     

Heterogeneity of macrophage populations in human lymphoid tissue and peripheral blood

Human lymphoid tissue and peripheral blood leukocytes were stained with six monoclonal antibodies directed against monocyte/macrophage populations. The staining pattern described by each of these monoclonal reagents was compared with the distribution of morphologically distinguishable tissue macrophages. The results show that there exists considerable heterogeneity of tissue macrophages based on the expression of surface and/or cytoplasmic antigens; furthermore, the distribution of cells bearing particular antigenic determinants is associated with distinct regions in normal lymphoid tissue. Double staining methods demonstrated that these antibodies bind to different, as well as to identical, macrophage populations. OKM-1 antibody binds predominantly to sinus histiocytes and tingible body macrophages. The Leu M-1 reagent stains interdigitating reticulum cells, while the KiM-4 antibody labels follicular dendritic cells. Leu M-3 antibody identifies cells predominantly in the germinal center, and histiocytes lining the sinuses. Both CM-1 and BRL-M.1 appear to stain tissue macrophages distributed throughout the lymphoid tissue.     

The effects of corticosteroid concentrations on lymphocyte blastogenesis

High concentrations of methylprednisolone added for prolonged periods in vitro to lymphocyte cell cultures inhibited allogeneic-cell-induced or phytohemagglutinin (PHA)-induced blastogenesis in contrast to lower concentrations which were inhibitory only if added before or within several hours of blastogenic induction.     

A defect of in vitro monocyte leukotaxis induced by cutaneous allograft rejection

Previous studies have demonstrated defective monocyte leukotaxis in human sarcoidosis and tuberculosis, diseases associated with chronic cellular immune activation. In the present study, we sought to determine if acute cellular immune activation might also alter this monocyte function, using a primate model of skin allograft rejection. A profound, short-lived depression of monocyte leukotactic responsiveness occurred during the time of allograft rejection. The leukotactic defect was mediated by a cell-directed inhibitor in plasma, a heat-stable protein of 230,000 daltons molecular weight. Neither monocyte leukotactic responsiveness nor plasma inhibitory activity was affected by autografting or nonspecific cutaneous inflammation. A physicochemically and functionally similar inhibitor was produced in vitro during the course of one-way primed mixed lymphocyte cultures. The cell-directed inhibitor appears to be a lymphocyte product which is clearly distinguishable from MIF and leukotactic lymphokines.     

DNA repair and replication in human fibroblasts treated with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

DNA repair and replication were examined in diploid human fibroblasts after treatment with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Unscheduled DNA synthesis exhibited a linear response to BPDE-I concentrations up to 1.5 μM and a saturation plateau after higher concentrations. Maximal unscheduled DNA synthesis was observed in the first hour after treatment with synthesis diminishing progressively thereafter. Half-maximal unscheduled DNA synthesis was seen within 4–6 h after treatment with 0.7 μM BPDE-I. DNA replication was inhibited by BPDE-I in a dose- and time-dependent fashion. The mechanisms of this inhibition were characterized by velocity sedimentation of pulse-labeled nascent DNA in alkaline sucrose gradients. Very low concentrations of BPDE-I (0.03 and 0.07 μM) were found to inhibit replicon initiation by up to 50% within 30–60 min after treatment. Recovery of initiation following these low concentrations was evident within 3 h after treatment. Higher concentrations of carcinogen inhibited DNA synthesis in active replicons. This effect was manifested by a reduction in incorporation of precursor into replication intermediates of greater than 1·10⁷ Da with the concurrent production of abnormally small nascent DNA. When viewed 45 min after treatment with 0.17 μM BPDE-I the combination of these two effects partially masked the inhibition of replicon initiation. However, even after treatment with 0.33 μM BPDE-I an effect on initiation was evident. These results reveal a pattern of response to BPDE-I that is quite similar to that produced by 254 nm radiation.     

Neuroendocrine response to cold in Raynaud's syndrome

Eleven patients with Raynaud's syndrome accompanied by monospecific IgG ANA, nine patients with Raynaud's syndrome in the absence of ANA, and nine normal volunteers were exposed to an ambient cold challenge during which time venous blood was continuously sampled. ANA negative patients were shown to have significantly higher levels of cortisol during a cold challenge than either ANA positive patients or normal controls, and exhibited significantly lower levels of plasma norepinephrine compared with normal controls. ANA positive patients did not differ significantly from normals in their neuroendocrine response to cold. It is suggested that the high plasma cortisol found in Raynaud's syndrome in the absence of ANA may be responsible for the vasospasticity in this group of patients.